Design of polyhedral oligomeric silsesquioxane (POSS) based thermo-responsive amphiphilic hybrid copolymers for thermally denatured protein protection applications

Abstract

A series of thermo-responsive amphiphilic hybrid copolymers with a random brush-like structure were synthesized by copolymerizing hydrophilic poly(ethylene glycol)methacrylate (PEGMA) and hydrophobic polyhedral oligomeric silsesquioxane methacrylate (POSSMA) together with temperature sensitive poly(propylene glycol)methacrylate (PPGMA) via atom transfer radical polymerization (ATRP). The resulting poly(PEGMA–PPGMA–POSSMA) (PEPS) hybrid copolymers show a lower critical solution temperature (LCST) in the range of 31–33 °C. Static and dynamic light scattering (SLS and DLS) studies show that micellar structures created by the PEPS copolymer in aqueous media were core-shell structures and possessed a thick hydration layer. The presence of a small amount of POSS (3.1 wt%) in the PEPS copolymers lowered the CMC of the micelles at room temperature by one order of magnitude compared to samples without POSSMA (PEP). Incorporation of POSSMA also enhanced the stability of the formed micelles, i.e. PEPS containing 6.7 wt% POSS exhibited a constant hydrodynamic radius, R_h (∼65 nm), and an aggregation number, N_agg (∼350), when the temperature was varied from 20 to 70 °C while PEP without POSS showed a large increase in both R_h and N_agg values. On the other hand, the change of R_g as the temperature increases could be attributed to the PPG brush adopting a more extended and compact conformation below and above LCST respectively. The thermo-responsiveness of the PPG brush in PEPS hybrid micelles was also exploited to mimic the natural GroEL–GroES chaperone functionalities for renaturation of thermally denatured proteins. Above LCST of PPG, the chaperone-like system comes into effect with hydrophobic PPG domains on the micelle surface, providing spontaneous capture and protection of the unfolded proteins, thus inhibiting the undesired protein aggregation at elevated temperatures. Upon cooling, PPG returns to its hydrophilic state, thereby inducing the release of the bound unfolded proteins. The renaturation process of the detached proteins is spontaneously accomplished by the presence of PEG and OH-groups in the micelle corona. The working mechanism and thermal denaturation protection effect were also investigated by DLS, SLS and circular dichroism (CD) spectroscopy. In the presence of PEPS hybrid micelles, the protection efficiencies for GFP, lipase and lysozyme that can be achieved during the heat-induced denaturation process are 81.4%, 89.3% and 88.7%, respectively. Cell culture and cytotoxicity studies revealed that the PEPS hybrid micelles could be effectively internalized by C6 glioma cells and possess good cell biocompatibility. These interesting findings open up new opportunities to exploit PEPS hybrid copolymers as artificial chaperones for protecting unfolded proteins from toxic aggregation at high temperatures.